ABI Innovation: Data Driven Model of Polymerase Activity

University Of Colorado At Boulder, Boulder CO

Investigators

Abstract

The majority of cells in an organism contain the same DNA, and yet they are able to develop along different paths to achieve different functions and phenotypes. The cell type identity is defined, at least in part, by the specific regions of the genome that are transcribed from DNA to RNA. Transcription is the basic cellular process that creates the RNA intermediate that either codes for proteins, mRNAs, or regulates other processes, producing various non-coding RNAs (ncRNAs) like enhancer RNAs (eRNAs). Both types of transcript cell differentiation paths or cellular responses to environmental influences. RNA polymerases are the enzymes that produce RNA transcripts, such as mRNAs and ncRNAs, and they exist in cells either as molecules that are maintain their association with the DNA template or are released being transported away from the DNA and acted on by other cellular machinery. One complication to analyzing the transcription process is that genes can overlap, so the presence of a polymerase and a nascent RNA transcript might indicate activity of either gene using current methodologies. Since RNA transcripts have a high propensity to fold into three dimensional shapes they that can affect the downstream processes, both sequence and shape of the nascent RNA are important characteristics of nascent transcripts still associated with DNA as well as the RNA transcripts that are released from the DNA. Only recently have high throughput nascent transcription assays become available; these allow researchers to assess the activity of cellular polymerases directly. This project seeks to develop an integrated analysis framework for nascent transcription data. Critically, changes in the shapes or levels of transcripts provide information as to how mutations, or other perturbations, have affected an RNA polymerase's functional properties. Understanding the detailed molecular basis of transcription is critical to many research areas including biochemistry, molecular biology and computational biology. As such, careful attention will be paid to the development of educational and computational resources to support of the larger community. The overall goal of this project is to develop of an integrated framework for the interpretation and analysis of nascent transcription. This project leverages a mathematical model of RNA polymerase II that, when fit to nascent transcription data, quantitatively characterizes not only the levels but also the shapes of all transcripts genome-wide. Specifically, this project has 4 overarching goals, as follows: Leverage patterns of differential nascent transcription to identify overlapping transcripts; detection of overlapping transcription at regulatory regions like enhancers may provide insight into how such ncRNAs function. Capture termination of transcription within a principled mathematical model of polymerase behavior; the algorithm developed here will inform on the conflicting hypotheses about how transcription termination occurs. Identify the impact of technical choices, such as protocol selection and sequencing depth, on the detection and characterization of transcripts, thereby recommending best practice in nascent transcription studies. Enable broader adoption of nascent transcription analysis through construction of an educational module that supports the use of these computational tools; the module will encompass the software, websites, and user documentation. In summary, the research proposed here will provide an unprecedented perspective on differences in transcription characteristics at individual genes and how perturbations affect them. By quantifying alterations in transcription at individual genes and their magnitude, the end result of this project will be an integrated framework for the interpretation of nascent transcription. Datasets and codebase links, with descriptions, may be found at http://dowell.colorado.edu/resources.html . This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.